The GABA transaminase GabT is required for full virulence of Ralstonia solanacearum in tomato

The utilization of plant gamma-aminobutyric acid (GABA) is essential for the pathogenicity of the bacterial plant pathogen Ralstonia solanacearum. A knockout mutant in the GABA transaminase-encoding gene gabT is unable to utilize GABA as a nutrient and its ability to cause disease in plants is strongly compromised. However, the proximity of the gabD gene (encoding a succinate semialdehyde dehydrogenase) in the same operon raises the question of whether an impact on the gabD gene underlies or contributes to the virulence attenuation of the ΔgabT mutant. In this work, we use genetic complementation to show that the expression of the gabT gene is able to rescue the impaired virulence of the ΔgabT knockout mutant in tomato plants, confirming that the gabT-encoded GABA transaminase is indeed required for full virulence of R. solanacearum in a natural host plant.

. Expression of gabT complements the virulence attenuation of the ΔgabT mutant: A. Predicted GABA catabolic pathway in R. solanacearum. GABA is converted into succinate semialdehyde (SSA) by GABA transaminases (GabT); SSA is used by succinate semialdehyde dehydrogenases (GabD) to generate succinate, which is eventually incorporated into the TCA cycle. B. Structure of the gabT-gabD operon in R. solanacearum GMI1000 (the gabT gene is also called goaG; Salanoubat et al., 2002). In the ΔgabT mutant, a Gentamycin (Gm) resistance cassette replaces the genomic sequence of the gabT gene. Arrows show the hybridization sites of the primers used in (C) for the validation of these strains. C. Validation of the ΔgabT mutant and ΔgabT/gabT complementation strains by PCR. Primers hybridizing in the gabT promoter and the gabT/gabD genes were used. The predicted size of the resulting fragment using primers a+c in a strain containing the original gabT allele (only present in the WT strain) is 2166 bp, while the predicted size of the fragment containing the Gm resistance cassette is 1760 bp. The predicted size of the resulting fragment using primers a+b (amplifying the gabT gene in both WT strain and complementation strain) is 1581 bp. Marker sizes are shown for reference. (D and E) Soil drenching inoculation assays in tomato Moneymaker plants using R. solanacearum GMI1000, a gabT mutant (ΔgabT), and a GabT complementation strain (ΔgabT/gabT). In (D) the results are represented as disease progression, showing the average wilting symptoms in a scale from 0 to 4. Values from 3 independent biological repeats were pooled together (mean ± SEM; n=36). (E) Survival analysis of the data in (D); the disease scoring was transformed into binary data with the following criteria: a disease index lower than 2 was defined as '0', while a disease index equal or higher than 2 was defined as '1' for each specific time point. Statistical analysis was performed using a Log-rank (Mantel-Cox) test. (F) Growth of R. solanacearum GMI1000 WT, ΔgabT, and ΔgabT/gabT in tomato stems. Five μL of bacterial suspension (10 6 cfu mL -1 ) were injected into the stem of 4-week-old tomato plants and xylem sap was extracted from each infected plant for bacterial quantification 2 or 3 days post-inoculation (dpi). Different colours represent values obtained in 3 independent biological repeats, and horizontal bars represent average values (n=6 plants per strain in each repeat). Statistical analysis was performed using a Student's t test. In E and F, asterisks indicate significant differences using the indicated statistical tests (ns, not significant, *** p<0.001, **** p<0.0001).

Description
Ralstonia solanacearum is the causal agent of bacterial wilt disease of more than 250 plant species, and is considered as one of the most destructive plant pathogens (Mansfield et al., 2012). R. solanacearum is a soil-borne bacterium that enters plants through the roots, eventually reaching the vascular system; bacterial proliferation in xylem vessels causes vascular blockage, which leads to plant wilting and death . We have recently shown that R. solanacearum promotes the production of gamma-aminobutyric acid (GABA) in plant cells, which in turn can be used as a nutrient to support bacterial replication and pathogenesis inside the plant . In other gram-negative bacteria, such as R. eutropha (also known as Cupriavidus necator) or Pseudomonas syringae, GABA catabolism involves GABA transaminases (GabT), which convert GABA into succinate semialdehyde (SSA); SSA is used by succinate semialdehyde dehydrogenases (SSA-DH / GabD) to generate succinate, which can be incorporated into the TCA cycle (Lutke-Eversloh & Steinbuchel, 1999;Park et al., 2010; Figure 1A). The genome of R. solanacearum contains a single copy of the gabT gene (RSc0029), which is located upstream of the gabD gene (RSc0028) in the same operon ( Figure 1B). Interestingly, a ΔgabT knockout mutant was unable to use GABA as a nutrient, and severely impaired in its ability to cause disease symptoms in the model plant Arabidopsis thaliana and to grow in stems of tomato plants, their natural host . These results suggest that R. solanacearum requires the GabT transaminase to cause disease. However, given that the gabT and gabD genes belong to the same operon, it is formally possible that the mutation in gabT affects the expression of gabD, which could also contribute to the reduced virulence observed in the ΔgabT mutant. In order to address this possibility, we complemented the ΔgabT mutant using an integrative plasmid that leads to the expression of the gabT gene driven by its own promoter (see methods; Figure  1C). First, we inoculated the ΔgabT mutant in tomato plants by soil-drenching, an inoculation method that mimics the natural infection route of R. solanacearum, and found that, as previously shown in Arabidopsis, the ΔgabT mutant is significantly impaired in its ability to cause disease symptoms in tomato, in comparison to the GMI1000 wild-type reference strain ( Figure  1D and 1E). Importantly, the symptoms induced by the complementation strain expressing gabT in the ΔgabT mutant background (ΔgabT/gabT) were similar to those induced by the GMI1000 wild-type strain ( Figure 1D and 1E). Moreover, upon injection in tomato stems, expression of the gabT gene was able to rescue the attenuation in growth observed in the ΔgabT mutant ( Figure 1F). Altogether, these results indicate that the virulence attenuation observed in the ΔgabT mutant  Figure 1D and 1E) is indeed caused by mutation of the gabT gene. These results also suggest that either the gabD gene is still expressed in the ΔgabT mutant, or the gabD gene is not required for virulence. However, if the gabD gene is not required for virulence, how does R. solanacearum catabolize SSA to complete the GABA catabolic pathway? In R. eutropha it has been shown that a gabD mutant still displays SSA-DH activity (Lutke-Eversloh & Steinbuchel, 1999), suggesting the presence of additional SSA-DH enzymes, and such non-GabD SSA-DH enzymes have been identified in other bacteria, such as Escherichia coli or Klebsiella pneumoniae (Marek & Henson, 1988;Sanchez et al., 1989). Similarly, even if the gabD gene were affected in the ΔgabT mutant, it is possible that R. solanacearum uses other SSA-DH enzymes to complete the GABA catabolic pathway in the ΔgabT/gabT complementation strain.

Generation of R. solanacearum gabT complementation strain
To complement gabT in the ΔgabT mutant , the gabT gene (RSc0029) with its promoter (300 bp upstream of ATG of the gabT gene) was amplified by PCR, cloned into pENTR-TOPO, and then introduced into pRCT-GWY (Monteiro et al., 2012) by LR reaction (in vitro recombination between the entry clone -pENTR-TOPO-and the destination vector -pRCT-GWY), resulting in the pRCT-pgabT-gabT (Vailleau et al., 2007) plasmid. The integrative pRCT-pgabT-gabT plasmid, triggering the expression of gabT under the control of the native gabT promoter, was transformed into the R. solanacearum ΔgabT mutant strain (in GMI1000 background) by natural transformation, leading to the stable integration of the pgabT-gabT fragment into a permissive site of the bacterial chromosome, as previously described (Monteiro et al., 2012). The complementation strain ΔgabT/gabT was selected using Tetracycline (10 μg mL -1 ) and confirmed by PCR using gabT specific primers ( Figure 1b and Table S1).

Pathogen inoculation assays
For R. solanacearum soil-drenching inoculation, 12 3-4 week-old tomato plants for each bacterial strain (grown in Jiffy pots) were inoculated by soil drenching with a bacterial suspension containing 10 8 colony-forming units per mL (CFU mL -1 ). 300 mL of inoculum of each strain was used to soak each set of plants. After 20-minute incubation with the bacterial inoculum, plants were transferred from the bacterial solution to a bed of potting mixture soil in a new tray (Vailleau et al., 2007). Scoring of visual disease symptoms on the basis of a scale ranging from '0' (no symptoms) to '4' (complete wilting) was performed as previously described (Vailleau et al., 2007).
Stem injection assays with R. solanacearum were performed as previously described Wang et al., 2021a;Wang et al., 2021b). Briefly, 5 μL of a 10 6 CFU mL -1 bacterial suspension were injected into the stems of 4-week-old tomato plants and 2.5 μL xylem sap were collected from each plant for bacterial quantification 2 or 3 days-post inoculation (dpi). Injections were performed 2 cm below the cotyledon emerging site in the stem, while the samples were taken at the cotyledon emerging site. CFUs were counted by spreading serial dilutions on solid BG medium (Plener et al., 2010).